A “grappling hook” interaction connects self-assembly and chaperone activity of Nucleophosmin 1

Abstract How the self-assembly of partially disordered proteins generates functional compartments in the cytoplasm and particularly in the nucleus is poorly understood. Nucleophosmin 1 (NPM1) is an abundant nucleolar protein that forms large oligomers and undergoes liquid–liquid phase separation by binding RNA or ribosomal proteins. It provides the scaffold for ribosome assembly but also prevents protein aggregation as part of the cellular stress response. Here, we use aggregation assays and native mass spectrometry (MS) to examine the relationship between the self-assembly and chaperone activity of NPM1. We find that oligomerization of full-length NPM1 modulates its ability to retard amyloid formation in vitro. Machine learning-based structure prediction and cryo-electron microscopy reveal fuzzy interactions between the acidic disordered region and the C-terminal nucleotide-binding domain, which cross-link NPM1 pentamers into partially disordered oligomers. The addition of basic peptides results in a tighter association within the oligomers, reducing their capacity to prevent amyloid formation. Together, our findings show that NPM1 uses a “grappling hook” mechanism to form a network-like structure that traps aggregation-prone proteins. Nucleolar proteins and RNAs simultaneously modulate the association strength and chaperone activity, suggesting a mechanism by which nucleolar composition regulates the chaperone activity of NPM1.

prepared in the absence and presence of varying molar ratios of NPM1 variants. To determine the effects of different NPM1 concentrations on the aggregation half time of Aβ42 fibril formation, fluorescence data was normalized and fitted to an empirical logistic5 function with an equation (1): where F0 is the baseline value, P the plateau value, x the time point value, x0 the time factor, h the Hill slope steepness factor, s the control factor. The aggregation half-time τ0 values were derived from the following equation (2): Where x0 is the time factor, h the Hill slope steepness factor, s the control factor. Data is represented as the average±standard deviation from 4-5 experiments.

Native mass spectrometry
All purified NPM1 variants were buffer-exchanged into 1 M ammonium acetate, pH 8.0, using Bio-Spin P-6 columns (BioRad, CA). Mass spectra were acquired on a Waters Synapt G1 travelling wave ion mobility mass spectrometer modified for high-mass analysis (MS Vision, NL) equipped with an offline nanospray source. The capillary voltage was 1.5 kV, the source pressure was 8 mbar, and the source temperature was 80°C. Mass spectra were visualized using MassLynx 4.1 (Waters, UK).

Negative stain electron microscopy
For negative stain EM, a final concentration of 3 μM Aβ42, 3 μM NPM1240-294, or 3 μM Aβ42 + 3 μM NPM1240-294 were prepared in 160 mM NaCl, 16 mM Tris, 4 mM NaPi, 0.04 mM EDTA pH 8 and incubated overnight at 37°C. The samples were then centrifuged at 17000 x g for 30 min and 80% of the supernatant was removed. The remainder was resuspended and 5 µL was loaded to 200 mesh copper grids with Formvar/Carbon support film that had been glow-discharged at 25 mA for 2 min. After one minute of incubation, the liquid was removed and the negative staining was carried out by applying a 5 µl of 1 % (w/v) uranyl acetate (in H2O) to the grid for 20-30 seconds. Then, the liquid was removed and the procedure was repeated 6 times 46 . The grids were imaged in Talos 120 C G2 (Thermo Scientific) equipped with a CETA-D detector.

cryo-EM data collection and processing
Four microliters of 0.75 mg/ml of purified NPM1 were applied to Cryomatrix 2/1 grids (glow discharged for 2 min at 25 mA) in a Vitrobot MK IV (Thermo Fisher Scientific) at 4°C and 100% humidity. Sample excess was removed by blotting for 4 s using a blot force of 1 followed by vitrification in liquid ethane.
The data were collected on a Krios G3i electron microscope (Thermo Fisher Scientific) at an operating voltage of 300 kV with Gatan BioQuantum K3 image filter/detector (operated with a 10 eV slit) at the Karolinska Institutet's 3D-EM facility, Stockholm, Sweden. The data were collected using EPU (Thermo Fisher Scientific). An EFTEM SA magnification of 165,000x was used, resulting in a pixel size of 0.505 Å, with a total dose of 54 e -/Å 2 divided across 60 frames over 2 s (fluency of 6.9 e-/px/sec). Target defocus values were set between -0.2 to -2 µm and using a stage tilt of 0° or -20°. The data processing strategy is schematized in Fig S3a. Motion correction and CTF estimation was performed in Warp 47 .The micrographs were then imported in Scipion3 48 and denoised using Janni for picking with crYOLO 49 using the pre-trained model for denoised micrographs. The particle picks were pruned using XMIPP Deep Micrograph Cleaner 50,51 . The particles were then extracted in WARP (64 px box, 2-fold binning to 2.02 Å/px). 2D classification of the particle set was performed in CryoSPARC v3.01 52 and the good 2D classes were manually selected for ab-initio reconstruction in CryoSPARC of 1 class. The particles were then refined in RELION 4.0 beta 53 .The refined particles were re-extracted in WARP (192 px box, 1.01 Å/px) and further refined in RELION 4.0 beta using a mask (covering N-terminal and C-terminal domains) and subjected to 3D classification without alignment (6 classes, T 20) using the same mask. The class with very weak density for C-terminal domains (Class 1) and the class with the strongest density for C-terminal domains (Class 6) were then selected and refined individually in CryoSPARC v3 non-uniform refinement. In order to improve the resolution on the Nterminal region and be able to fit an atomic model for this region, Class 6 was further subjected to 3D classification in RELION 4.0 beta. The two classes which reconstructed to the highest resolution were pooled and re-extracted in WARP (382 px box, 0.505 Å/px). These particles (106,905 particles) were finally refined and post-processed in RELION 4.0 with a mask on the N-terminal region. The resulting map was used for the refinement of an atomic model for the N-terminal region. The PDB 5EHD, which was used as a starting model, was docked in the map and refined in Coot 0.9.8.1 54 and REFMAC5 55 .

Binding energy calculations
The peptide: protein binding energy was computed using MM/GBSA (Molecular Mechanics / Generalized Born Surface Area) method 56 as obtained from the following equations: Ebon is composed of three bonded (bond, angle and dihedral) energy terms. Evdw and Eelec are the van der waals and electrostatic non-bonded interaction components respectively. These energies are calculated using Molecular Mechanics (MM) force field expressions of AMBER. Gpol is the polar solvation energy which is obtained by solving the Generalized Born (GB) solvation model. Gnpol is the non-polar solvation energy estimated through an empirical linear relationship (γ*SASA), where "γ" is the surface-tension and "SASA" is the Solvent Accessible Surface Area. The complex state structures were used to generate the corresponding free states of the protein and peptide. The continuum solvent environment was represented with an implicit GB model (IGB=2). The internal dielectric constant for the protein/peptide and the solvent was set to 1 and 80 respectively, γ= 0.0072 kcal/mol/Å 2 and the salt-concentration was set to 0.15 mM. The MMPBSA.py script 57 available through the AMBER18 suite of programs was used to carry out the calculations.

Light microscopy and fluorescence microscopy
Unlabeled NPM1 at 10 μM in 150 mM NaCl, 2 mM DTT, in 10 mM Tris pH 7.5 with or without basic peptides at concentrations of 10 or 100 μM were added to Corning 3651 polystyrene 96-well plates (Corning, USA). For light microscopy imaging, the mixtures were incubated at room temperature for 1 h and the liquid-liquid phase separation images in brightfield mode were recorded using a Zeiss Cell Observer microscopy instrument (Carl Zeiss AG, Germany) at a final magnification of 400x using LD Plan Neofluor 0.6 Corr Air objective. Images were recorded from initial focus point ±40 μm with a step of 1 μm.
For DroProbe imaging, 20 µM of 1,2-bis[4-(3-sulfonatopropoxyl) phenyl]-1,2diphenylethene (DroProbe, AIEgen Biotech) was added to NPM1 in an 18 well chamber slide and incubated at room temperature for 5 min. Fluorescence microscopy images were acquired using a Nikon Eclipse Ti series inverted microscope (Nikon) equipped with Crest X-light V2 series confocal unit (Nikon), using 395 nm excitation wavelength and 3% laser power. Images were acquired using an S Plan ELWD 60X/0,70 oil immersion objective (Nikon) and a Zyla sCMOS camera (Andor        The NTD pentamer is rendered as electrostatic surface, the SURF6 peptide is shown as ribbon in turquoise with basic residues as sticks. (f) T1/2 of Ab42 aggregation curves reveal that SURF6299-326 has no pronounced effect on Ab42 aggregation alone, but significantly reduces the chaperone-like activity of FL NPM1. Error bars indicate the standard deviation of n=4 repeats. Significance was calculated using Student's T-Test for paired samples with equal variance. Figure S8. AF models for 29 homologs of NPM1 with identical domain architectures. Models are colored from N-to C-terminus and only non-disordered residues of the CTDs that are within a 4Å distance cut-off from the NTD are shown.